Fig. 2: DLD and the E3 binding protein of PDH are likely modified by HNO. | Nature Communications

Fig. 2: DLD and the E3 binding protein of PDH are likely modified by HNO.

From: Pyruvate dehydrogenase operates as an intramolecular nitroxyl generator during macrophage metabolic reprogramming

Fig. 2

a Schematic illustration of atom transitions using uniformly labeled 13C-Leucine ([U-13C]) (labeled carbons in blue) as tracer for determination of mass isotopologue distributions to infer relative intracellular fluxes. BCAT Branched-chain amino acid aminotransferase, BCKDH Branched-chain α-keto acid dehydrogenase. b, c WT and Nos2 −/− BMDMs from male and female mice (mixed sex in all groups) were activated for 16 h with LPS + IFNγ and cultured 4 h with labeled tracer. Bars show carbon incorporation into M + 0–6 isotopologues of indicated intermediates (n = 4 mice per genotype). Data were analyzed by two-way ANOVA with Sidak’s post-tests. d DLD in-gel activity and native IB of mitochondrial fractions from WT BMDMs untreated or 16 h-stimulated with LPS + IFNγ (n = 9 total mice). Treatments with DETA/NO/DTT/lipoate were performed post harvest on lysates. e Mechanism of generation of HNO. f Outputs of reaction of HNO with common thiols. g DLD in-gel activity of mitochondrial fractions from WT BMDMs stimulated for 16 h with LPS + IFNγ and 4 h with AS. Bar graphs show quantified diaphorase activity (n = 3 technical repeats). h DLD dehydrogenase activity in mitochondrial lysates from WT after 16 h-stimulation with LPS + IFNγ or either 400μM or 5 mM AS or DEA/NO for 4 h (n > 3 mice). i Lysates from  (h) were incubated 1 h at 37 °C with reducing agents (10 mM) before assessment of DLD dehydrogenase activity. Basal values of activity of LPS + IFNγ, AS and DEA/NO from (h) were set to 1 (n > 3 mice). Data in (g–i) were analyzed by one-way ANOVA with Dunnet’s multiple comparisons test. j Schematic illustration of PDH enzyme with emphasis on DLD and E3bp. k E3bp-IB on native gels of mitochondrial fractions from BMDMs after 16 h stimulation with LPS + IFNγ or DETA/NO (500 μM), or with AS or DEA/NO for 4 h. Lysates pre-native PAGE were incubated with 10 mM DTT for 1 h at 37 °C (n = 8 total mice). All error bars display mean ± SEM. Data shown are representative of two or more independent experiments. p values > 0.05 are reported as “ns” unless otherwise specified. Source data are provided as a Source Data file.

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